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. 2017 Sep 29;7:12409. doi: 10.1038/s41598-017-12635-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Effect of MB on mitochondrial ROS production, phagocytosis, caspase-1 activity, and NLRP3 promoter activity. (A) BMDMs were treated with rotenone (160 μM) and the indicated dosages of MB for 6 h, after which mitochondrial ROS generation was analyzed. (B) LPS-primed BMDMs were treated with rotenone with/without MB, and secretion of IL-1β was measured by ELISA. (C) BMDMs were incubated with two different diameters (30 nm or 1 μm) of fluorescent beads for 6 h, followed by measurement of intracellular beads based on florescence intensity. (D) Recombinant human caspase-1 (rhCasp1) was incubated with its substrate (YVAD-pNA) in the presence of MB as indicated. (E) Luciferase-expressing plasmids (pGL3) controlled by mouse NLRP3 promoters (−1,327 nt to + 166 nt or −1,216 nt to + 166 nt) were transfected into RAW 264.7 cells, which were treated with/without LPS. Left schematic figures indicate the mouse NLRP3 promoter regions (filled boxes, NF-κB binding sites; LUC, luciferase gene). (F) RAW 264.7 cells were transfected with pGL3 (−1,327 nt to + 166 nt) and treated with MB and/or LPS. Relative luciferase activity (RLA) was analyzed. Bar graph presents the mean ± SD.